Spectroscopic studies at the single-molecule or single-particle levels have provided invaluable information on the dynamics of complex systems in fields as different as materials science and molecular cell biology. These measurements eliminate the confounding ensemble average and provide direct observation of heterogeneity in space and time. To study long-term dynamics, a confocal or total internal reflection detection scheme has been used typically in conjunction with immobilized molecular or particle probes. This widely used experimental scheme, however, does not provide a direct correlation between local dynamics and the position of the molecule or particle in three dimensions, which can be a critical in understanding the link between microscopic processes and macroscopic phenomena.
There have been several proposed designs for following a particle in 3D non-invasively. In one example, an overfilled detector was used to detect the z position, and a position sensor was used to detect the x and y positions. The method relies on a lensing effect of a diffusive polystyrene sphere. A limit to the size of the bead is set by the diffraction limit of the light source and the difference in index of refraction between the bead and its surrounding medium. In another design, a target particle was excited with a rotating beam and the 3D position of the target was determined by off-line demodulating of the fluorescence signal. The resulting time resolution of 30-60 ms is dependent on the brightness of the particle and the integration time. Other methods that actively trap small particles or single molecules have also been demonstrated using, for instance, optical tweezers and two-dimensional electrical fields.
The program of a cell is encoded in the genes and executed by protein molecules. Genetic miscoding and aberrant folding of proteins, respectively, cause diseases such as sickle cell anemia and the neural degenerative Alzheimer's disease. Proteins in living cells exhibit motions that may be confined, directed, or diffusive. For example, with the use of fluorescent recovery after bleach (FRAP), the diffusion constant of green fluorescent proteins has been found to decrease from 87 μm2s−1 in water to 25 μm2s−1 in cytoplasm, to 20-30 μm2s−1 in the mitochondrial matrix, and to 5-10 μm2s−1 in the endoplasmic-reticulum lumen. These motions are often coupled to biochemical events involving the protein molecules that regulate cellular processes, such as sensing and response to stimulants and/or stress in the environment.
Single-molecule spectroscopy (SMS) is a uniquely powerful technique that affords close examination of the time trajectory and reactions of individual molecules that are otherwise hidden in the ensemble average. It is extremely difficult, if not impossible, using conventional spectroscopy, to observe the sequence of events in processes such as conformational dynamics of protein molecules and intermolecular interactions between enzymes and substrates. The dynamic configuration of biological macromolecules has direct implications on their time-dependent reactivity. Statistical analysis can be used to deduce dynamic configurations once the time dependence of the fluorescence intensity of biological labels is known. With SMS, in-depth understanding of the activities of individual biomolecules is possible, which is essential for the study of rare events that are hidden in the broad spatial-temporal distribution of a bulk experiment.
To date, most studies of single biological molecules have been conducted in vitro where the conditions are markedly different from those in a live cell or in a physiological system. Herein is disclosed apparatus and methods that make it possible to use SMS in a live-cell and in other complex environments.